Vincenzo Visco

Chloroquine enhances human CD8+ T cell responses against soluble antigens in vivo

The presentation of exogenous protein antigens in a major histocompatibility complex class I–restricted fashion to CD8+ T cells is called cross-presentation. We demonstrate that cross-presentation of soluble viral antigens (derived from hepatitis C virus [HCV], hepatitis B virus [HBV], or human immunodeficiency virus) to specific CD8+ T cell clones is dramatically improved when antigen-presenting dendritic cells (DCs) are pulsed with the antigen in the presence of chloroquine or ammonium chloride, which reduce acidification of the endocytic system. The export of soluble antigen into the cytosol is considerably higher in chloroquine-treated than in untreated DCs, as detected by confocal microscopy of cultured cells and Western blot analysis comparing endocytic and cytosolic fractions. To pursue our findings in an in vivo setting, we boosted groups of HBV vaccine responder individuals with a further dose of hepatitis B envelope protein vaccine with or without a single dose of chloroquine. Although all individuals showed a boost in antibody titers to HBV, six of nine individuals who were administered chloroquine showed a substantial CD8+ T cell response to HBV antigen, whereas zero of eight without chloroquine lacked a CD8 response. Our results suggest that chloroquine treatment improves CD8 immunity during vaccination.

Immune responses to all ErbB family receptors detectable in serum of cancer patients

Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immuno-histochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors.

Epstein-Barr virus latent membrane protein 1 promotes concentration in multivesicular bodies of fibroblast growth factor 2 and its release through exosomes

FGF‐2, a potent angiogenic factor that is involved in tumor invasion, is known to be released extracellularly by a nonclassical secretory pathway. Recently it has become clear that Epstein‐Barr virus, specifically its oncoprotein LMP1, can induce expression of angiogenic factors. Among these factors is FGF‐2. LMP1 not only promotes expression of FGF‐2, but also the release extracellularly of its 18‐kDa isoform. We analyzed the mechanism of FGF‐2 release induced by LMP1. Confocal immunofluorescence microscopy revealed colocalization of FGF‐2 with LMP1 in small dots also stained positively for CD63 and cathepsin D, markers of late endosomes or multivesicular bodies. Biochemical analysis and immunoelectron microscopy of purified exosomal fractions from cotransfected cells demonstrated increased release of exosomes and the concentration of LMP1 and FGF‐2 in these structures. Moreover, cotransfection appeared to induce partial redistribution of the Na+/K+‐ATPase, which participates in FGF‐2 release, from the plasma membrane to the intracellular LMP1/FGF‐2 positive dots. Treatment with ouabain, which inhibits Na+/K+‐ATPase activity, partially suppressed FGF‐2 secretion via exosomes in a dose‐dependent manner. The results suggest that exosomes may represent a previously unrecognized mechanism for FGF‐2 release mediated by LMP1, and that this pathway involves the activity of Na+/K+‐ATPase.

Extracorporeal shock wave therapy promotes cell proliferation and collagen synthesis of primary cultured human tenocytes

PurposeThe aim of this study was to investigate whether the effects of extracorporeal shock wave therapy (ESWT) could affect the behavior of primary cultured human tenocytes over a 12-day period.MethodsIn this controlled laboratory study, primary human tenocytes were established from semitendinosus tendons collected from 3 patients undergoing arthroscopic anterior cruciate ligament (ACL) reconstruction. Cell viability, overall cell morphology, cell proliferation, and collagen synthesis following ESWT have been evaluated.ResultsESWT significantly interferes with the overall cell morphology, by impairing dedifferentiation of the cells. Furthermore, a shock wave-mediated growth-promoting effect was measured by the MTT (tetrazolium) colorimetric assay and by the proliferation marker Ki67. Lastly, a significant increase in collagen (mainly type I) synthesis by ESWT-tenocytes compared with control cells was found.ConclusionsShock wave treatment promoted cell growth and collagen synthesis of primary cultured human tenocytes. The clinical benefits of ESWT may be ascribed to an increased efficiency of tendon repair after injury.

Differential response to keratinocyte growth factor receptor and epidermal growth factor receptor ligands of proliferating and differentiating intestinal epithelial cells

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco‐2 cells upon confluence‐induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up‐modulated in post‐confluent differentiated cultures compared with the pre‐confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up‐regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF‐10, whereas they were not stimulated by the EGFR ligands TGFα and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post‐confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up‐modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

ErbB2 Immune Response in Breast Cancer Patients with Soluble Receptor Ectodomain

Investigation of ErbB2 immunity in human breast cancer employing recombinant expression sources in immunoblot analysis revealed ErbB2-specific antibodies of the IgG isotype in sera of 14 of 71 cancer patients and 1 of 31 normal donors. Reactivity was confirmed on ErbB2-specific immunoprecipitates. Independent evidence of existing ErbB2 immunity was obtained after in vitro transformation of peripheral blood leukocytes from six positive patients. Furthermore, in vitro immortalization of B-lymphocytes unmasked existent ErbB2 immunity in 1 of 8 patients negative for ErbB2 serum antibodies. Determining shed ErbB2 extracellular domain as an indirect measure of tumor burden in ErbB2-positive malignancy, elevated serum levels were observed in 16 of 71 breast cancer and 1 of 31 normal donor sera. Strikingly, existing ErbB2 immunity correlated significantly with elevated shed ErbB2 ectodomain among the patients analyzed. Incidence of both ErbB2 immunity and elevated ErbB2 extracellular domain increased with a progressed disease stage and was significantly associated with metastatic breast cancer. These observations implicate soluble ErbB2 amounts in vivo in the development of ErbB2 immunity in breast cancer. They further project serum analysis of ErbB2 immunity and soluble ectodomain as potential markers of disease progression in ErbB2-positive malignancy.

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long‐lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl‐2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down‐regulated in comparison with fibroblasts from normal lungs while the anti‐apoptotic protein Bcl‐2 expression was over‐expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl‐2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl‐2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction. J. Cell. Physiol. 228: 1516–1524, 2013.

Human colon fibroblasts induce differentiation and proliferation of intestinal epithelial cells through the direct paracrine action of keratinocyte growth factor

The effects exerted by the keratinocyte growth factor (KGF) on intestinal epithelial cells cultured in vitro are influenced by cell confluence and differentiation through the modulation of keratinocyte growth factor receptor (KGFR) expression. In order to better define the contribution of KGF on the intestinal epithelial cell differentiation and proliferation, here we developed a coculture model, able to mimick in vitro the epithelial–mesenchymal interactions of the bowel. In consequence of its ability to produce KGF, demonstrated by real‐time PCR and Western blot analysis, the human colon fibroblast cell line CCD‐18 has been selected as coculture partner for the intestinal epithelial Caco‐2 cell line. Analysis of the expression of the differentiation and proliferation markers CEA and Ki67, through double immunofluorescence assays, showed that either the coculture with CCD‐18 cells or the incubation with primary colon fibroblast‐derived conditioned media (CM‐F and CM‐F2) induced an increase in differentiation and proliferation of confluent intestinal epithelial Caco‐2 or HT29 cells, parallel to that obtained by KGF treatment. Use of anti‐KGF blocking antibodies and of a tyrosine kinase KGFR inhibitor demonstrated the contribution of KGF and the direct role of its receptor in the regulation of epithelial growth and differentiation, indicating that KGF is a crucial paracrine factor involved in promoting these effects. J. Cell. Physiol. 220: 204–213, 2009.

Extracorporeal Shock Wave Treatment (ESWT) Improves In Vitro Functional Activities of Ruptured Human Tendon-Derived Tenocytes

In vitro models of human tenocytes derived from healthy as well as from ruptured tendons were established, characterized and used at very early passage (P1) to evaluate the effects of Extracorporeal Shock Wave Treatment (ESWT). The molecular analysis of traditional tenocytic markers, including Scleraxis (Scx), Tenomodulin (Tnm), Tenascin-C (Tn-C) and Type I and III Collagens (Col I and Col III), permitted us to detect in our samples the simultaneous expression of all these genes and allowed us to compare their levels of expression in relationship to the source of the cells and treatments. In untreated conditions, higher molecular levels of Scx and Col I in tenocytes from pathological compared to healthy samples have been detected, suggesting – in the cells from injured tendon – the natural trigger of an early differentiation and repairing program, which depends by Scx and requires an increase in collagen expression. When ESWT (at the dose of 0.14 mJ/mm2) was applied to cultured tenocytes explanted from injured source, Scx and Col I were significantly diminished compared to healthy counterpart, indicating that such natural trigger maybe delayed by the treatment, in order to promote cellular repair. Herein, we show for the first time that ESWT enhances in vitro functional activities of ruptured tendon-derived tenocytes, such as proliferation and migration, which could probably contributes to tendon healing in vivo.

Phosphorylation-independent membrane relocalization of ezrin following association with Dbl in vivo

Ezrin, a widespread protein involved in cell migration, morphogenesis and cell adhesion, belongs to a large family of proteins known as ERM (ezrin, radixin, moesin). These three closely related proteins are thought to function as linkers between plasma membrane and actin cytoskeleton and their function is regulated by the small GTP-binding protein Rho. It has been previously shown that the active form of radixin can bind in vitro to Dbl, a Rho-specific guanine nucleotide exchange factor, although an in vivo interaction has not yet been demonstrated. In this paper, we attempted to investigate whether ezrin can also associate with Dbl. We show here that Dbl protein can effectively bind both in vitro and in vivo to the N-terminal region (amino acids 1–531) of a constitutively active mutant of ezrin and with the full-length molecule. We found that this binding is mediated by the Dbl pleckstrin homology domain, responsible for the proper subcellular localization of the Dbl protein. Moreover, we show that Dbl induces localization to the plasma membrane of both the active deletion mutant and the full-length ezrin proteins. Finally, we show that the relocalization of ezrin is independent of Dbl GEF activity. These results indicate that Dbl could induce translocation of ezrin to the plasma membrane through a mechanism that does not require ezrin C-terminus phosphorylation by Rho-associated kinases.